Targeted delivery of the probiotic Saccharomyces boulardii to the extracellular matrix enhances gut residence time and recovery in murine colitis.

Probiotic and engineered microbe-based therapeutics are an emerging class of pharmaceutical agents. They represent a promising strategy for treating various chronic and inflammatory conditions by interacting with the host immune system and/or delivering therapeutic molecules. Here, we engineered a targeted probiotic yeast platform wherein Saccharomyces boulardii is designed to bind to abundant extracellular matrix proteins found within inflammatory lesions of the gastrointestinal tract through tunable antibody surface display. This approach enabled an additional 24-48 h of probiotic gut residence time compared to controls and 100-fold increased probiotic concentrations within the colon in preclinical models of ulcerative colitis in female mice. As a result, pharmacodynamic parameters including colon length, colonic cytokine expression profiles, and histological inflammation scores were robustly improved and restored back to healthy levels. Overall, these studies highlight the potential for targeted microbial therapeutics as a potential oral dosage form for the treatment of inflammatory bowel diseases.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Evaluation of Surface Modified Live Biotherapeutic Products for Oral Delivery.

Live biotherapeutic products (LBPs), including symbiotic and genetically engineered bacteria, are a promising class of emerging therapeutics that are widely investigated both preclinically and clinically for their oral delivery to the gastrointestinal (GI) tract. One emergent delivery strategy involves the direct functionalization of LBP surfaces through noncovalent or covalent modifications to control LBP interactions with the GI microenvironment, thereby improving their viability, attachment, or therapeutic effect. However, unlike other therapeutic modalities, LBPs are living organisms which present two unique challenges for surface modifications: (1) this approach can directly interfere with key LBP biological processes (e.g., colonization, metabolite secretion) and (2) modification can be variable due to the dynamic nature of LBP surfaces. Collectively, these factors remain uncharacterized as they relate to the oral delivery of LBPs. Herein, we leverage our previously reported surface modification platform, which enables LBP surface-presentation of targeting ligands, to broadly evaluate and characterize surface modifications on LBPs. Specifically, we evaluate how LBP growth affects the dilution of surface-presented targeting ligands and the subsequent loss of specific target attachment over time. Next, we describe key surface modification parameters (e.g., concentration, residence time) that can be optimized to facilitate LBP target attachment. We then characterize how bioconjugation influences the suitability of LBPs for oral delivery by evaluating their growth, viability, storage, toxicity against mammalian cells, and in vivo colonization. Broadly, we describe key parameters that influence the performance of surface modified LBPs and subsequently outline an experimental pipeline for characterizing and evaluating their suitability for oral delivery.

Enhanced stability and clinical absorption of a form of encapsulated vitamin A for food fortification.

Food fortification is an effective strategy to address vitamin A (VitA) deficiency, which is the leading cause of childhood blindness and drastically increases mortality from severe infections. However, VitA food fortification remains challenging due to significant degradation during storage and cooking. We utilized an FDA-approved, thermostable, and pH-responsive basic methacrylate copolymer (BMC) to encapsulate and stabilize VitA in microparticles (MPs). Encapsulation of VitA in VitA-BMC MPs greatly improved stability during simulated cooking conditions and long-term storage. VitA absorption was nine times greater from cooked MPs than from cooked free VitA in rats. In a randomized controlled cross-over study in healthy premenopausal women, VitA was readily released from MPs after consumption and had a similar absorption profile to free VitA. This VitA encapsulation technology will enable global food fortification strategies toward eliminating VitA deficiency.

Viral vector-based gene therapies in the clinic.

Gene therapies are currently one of the most investigated therapeutic modalities in both the preclinical and clinical settings and have shown promise in treating a diverse spectrum of diseases. Gene therapies aim at introducing a gene material in target cells and represent a promising approach to cure diseases that were thought to be incurable by conventional modalities. In many cases, a gene therapy requires a vector to deliver gene therapeutics into target cells; viral vectors are among the most widely studied vectors owing to their distinguished advantages such as outstanding transduction efficiency. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes with many products approved for treating a range of diseases including cancer, infectious diseases and monogenic diseases. In addition, a number of active clinical trials are underway to further expand their therapeutic potential. In this review, we highlight the diversity of viral vectors, review approved products, and discuss the current clinical landscape of in vivo viral vector-based gene therapies. We have reviewed 13 approved products and their clinical applications. We have also analyzed more than 200 active trials based on various viral vectors and discussed their respective therapeutic applications. Moreover, we provide a critical analysis of the major translational challenges for in vivo viral vector-based gene therapies and discuss possible strategies to address the same.

Discovery and delivery strategies for engineered live biotherapeutic products.

Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy.

Polymer and Crosslinker Content Influences Performance of Encapsulated Live Biotherapeutic Products.

INTRODUCTION: Live biotherapeutic products (LBPs), or therapeutic microbes, are an emerging therapeutic modality for prevention and treatment of gastrointestinal diseases. Since LBPs are living, they are uniquely sensitive to external stresses (e.g., oxygen, acid) encountered during manufacturing, storage, and delivery. Here, we systematically evaluate how polymer and crosslinker concentration affects the performance of an encapsulated LBP toward developing a comprehensive framework for the characterization and optimization of LBP delivery systems. METHODS: We encapsulate a model LBP, Lactobacillus casei ATCC 393, in calcium chloride (CaCl2)-crosslinked alginate beads, and evaluate how alginate and CaCl2 concentrations influence LBP formulation performance, including: (i) encapsulation efficiency, (ii) shrinkage upon drying, (iii) survival upon lyophilization, (iv) acid resistance, (v) release, and (vi) metabolite secretion. Approaches from microbiology (e.g., colony forming unit enumeration), materials science (e.g., scanning electron microscopy), and pharmaceutical sciences (e.g., release assays) are employed. RESULTS: LBP-encapsulating alginate beads were systematically evaluated as a function of alginate and CaCl2 concentrations. Specifically: (i) encapsulation efficiency of all formulations was >50%, (ii) all alginate beads shrunk (after lyophilization) and recovered (after rehydration) similarly, (iii) at 10% alginate concentration, lower CaCl2 concentration decreased survival upon lyophilization, (iv) 10% alginate improved acid resistance, (v) sustained release was enabled by increasing alginate and CaCl2 concentrations, and (vi) encapsulation did not impair secretion of l-lactate as compared to free LBP. CONCLUSIONS: This research demonstrates that polymer content and crosslinking extent modulate the performance of polymer-based LBP delivery systems, motivating research into the optimization of material properties for LBP delivery systems.

Nanoparticles in the clinic: An update post COVID-19 vaccines.

Nanoparticles are used in the clinic to treat cancer, resolve mineral deficiencies, image tissues, and facilitate vaccination. As a modular technology, nanoparticles combine diagnostic agents or therapeutics (e.g., elements, small molecules, biologics), synthetic materials (e.g., polymers), and biological molecules (e.g., antibodies, peptides, lipids). Leveraging these parameters, nanoparticles can be designed and tuned to navigate biological microenvironments, negotiate biological barriers, and deliver therapeutics or diagnostic agents to specific cells and tissues in the body. Recently, with the Emergency Use Authorization of the COVID-19 lipid nanoparticle vaccines, the advantages and potential of nanoparticles as a delivery vehicle have been displayed at the forefront of biotechnology. Here, we provide a 5-year status update on our original "Nanoparticles in the Clinic" review (also a 2-year update on our second "Nanoparticles in the Clinic" review) by discussing recent nanoparticle delivery system approvals, highlighting new clinical trials, and providing an update on the previously highlighted clinical trials.

Enhanced Storage of Anaerobic Bacteria through Polymeric Encapsulation.

Live microbes such as lactobacilli have long been used as probiotic supplements and, more recently, have been explored as live biotherapeutic products with the potential to treat a range of conditions. Among these microbes is a category of anaerobes that possess therapeutic potential while exhibiting unique oxygen sensitivity and thus requiring careful considerations in the formulation and storage processes. Existing microbial formulation development has focused on facultative anaerobes with natural oxygen tolerance; a few strategies have been reported for anaerobes with demonstrated oxygen intolerance, warranting novel approaches toward addressing the challenges for these oxygen-sensitive anaerobes. Here, we develop a polymeric encapsulation system for the formulation and storage of Bifidobacterium adolescentis (B. adolescentis), a model anaerobe that loses viability in aerobic incubation at 37 °C within 1 day. We discover that this strain remains viable under aerobic conditions for 14 days at 4 °C, enabling formulation development such as solution casting and air drying in an aerobic environment. Next, through a systematic selection of polymer encapsulants and excipients, we show that encapsulation with poly(vinyl alcohol) (PVA) acts as an oxygen barrier and facilitates long-term storage of B. adolescentis, which is partially attributed to reduced generation of reactive oxygen species. Lastly, PVA-based formulations can produce oral capsule-loaded films and edible gummy bears, demonstrating its compatibility with both pharmaceutical and food dosage forms.

Cell therapies in the clinic.

Cell therapies have emerged as a promising therapeutic modality with the potential to treat and even cure a diverse array of diseases. Cell therapies offer unique clinical and therapeutic advantages over conventional small molecules and the growing number of biologics. Particularly, living cells can simultaneously and dynamically perform complex biological functions in ways that conventional drugs cannot; cell therapies have expanded the spectrum of available therapeutic options to include key cellular functions and processes. As such, cell therapies are currently one of the most investigated therapeutic modalities in both preclinical and clinical settings, with many products having been approved and many more under active clinical investigation. Here, we highlight the diversity and key advantages of cell therapies and discuss their current clinical advances. In particular, we review 28 globally approved cell therapy products and their clinical use. We also analyze >1700 current active clinical trials of cell therapies, with an emphasis on discussing their therapeutic applications. Finally, we critically discuss the major biological, manufacturing, and regulatory challenges associated with the clinical translation of cell therapies.

Modulating Oral Delivery and Gastrointestinal Kinetics of Recombinant Proteins via Engineered Fungi.

A new modality in microbe-mediated drug delivery has recently emerged wherein genetically engineered microbes are used to locally deliver recombinant therapeutic proteins to the gastrointestinal tract. These engineered microbes are often referred to as live biotherapeutic products (LBPs). Despite advanced genetic engineering and recombinant protein expression approaches, little is known on how to control the spatiotemporal dynamics of LBPs and their secreted therapeutics within the gastrointestinal tract. To date, the fundamental pharmacokinetic analyses for microbe-mediated drug delivery systems have not been described. Here, we explore the pharmacokinetics of an engineered, model protein-secreting Saccharomyces cerevisiae, which serves as an ideal organism for the oral delivery of complex, post-translationally modified proteins. We establish three methods to modulate the pharmacokinetics of an engineered, recombinant protein-secreting fungi system: (i) altering oral dose of engineered fungi, (ii) co-administering antibiotics, and (iii) altering recombinant protein secretion titer. Our findings establish the fundamental pharmacokinetics which will be essential in controlling downstream therapeutic response for this new delivery modality.

The evolution of commercial drug delivery technologies.

Drug delivery technologies have enabled the development of many pharmaceutical products that improve patient health by enhancing the delivery of a therapeutic to its target site, minimizing off-target accumulation and facilitating patient compliance. As therapeutic modalities expanded beyond small molecules to include nucleic acids, peptides, proteins and antibodies, drug delivery technologies were adapted to address the challenges that emerged. In this Review Article, we discuss seminal approaches that led to the development of successful therapeutic products involving small molecules and macromolecules, identify three drug delivery paradigms that form the basis of contemporary drug delivery and discuss how they have aided the initial clinical successes of each class of therapeutic. We also outline how the paradigms will contribute to the delivery of live-cell therapies.

Batch Culture Formulation of Live Biotherapeutic Products.

Live biotherapeutic products (LBPs) are an emerging therapeutic modality that are clinically investigated for treating pathogenic infections and inflammatory diseases. A major class of LBPs are feces derived microbial consortiums which require numerous process development steps (e.g. separation, purification, blending) to facilitate LBP formulation into oral dosage forms. A subset of these LBPs circumvent the need for continuous fecal processing by batch culture for individual strains of microbes that are rationally defined and combined in the final LBP formulation. Separately, delivery formulations (e.g. polymer encapsulation) are being developed for LBPs to improve storage and intestinal engraftment; however, formulation requires additional manufacturing processes distinct from fecal processing or batch culture. Here, a streamlined approach termed batch culture formulation (BCF) is developed to combine the individual batch culture and formulation processes into a single-step process. Based on a previously described polymeric film formulation that encapsulates LBPs, BCF is shown to reduce the number of required processes to formulate LBP-films without altering LBP phenotype, function, or storage profiles compared to the standard LBP-film formulation approach. Additionally, it is demonstrated that BCF facilitates scaled-fabrication from the milligram to gram scale with predictable loading, highlighting the potential that BCF has for clinical translation.

Coupled influences of particle shape, surface property and flow hydrodynamics on rod-shaped colloid transport in porous media.

HYPOTHESIS: Natural or engineered colloidal particles are often non-spherical in shape. In contrast to the widely-used "homogeneous sphere" assumption, the non-spherical particle shape is expected to alter particle-fluid-surface interactions, which in turn affect particle transport and retention. EXPERIMENTS AND SIMULATIONS: Polystyrene microspheres were stretched to rod-shaped particles of two aspect ratios (2:1, 6:1). The transport and retention behaviors of rods versus spheres were investigated in packed quartz sand columns and impinging jet systems. In parallel, a 3D trajectory model was employed to simulate particle translation and rotation, and to elucidate the role and underlying mechanisms of particle shape impact on transport. FINDINGS: Rods were observed to undergo rotating and tumbling motions in response to fluid shear from experiments and simulations. However, no distinct retention trends between rods and spheres were observed from column studies, despite BSA-coating on particles, Fe-coating on sand or velocity change. This was primarily due to the super-hydrophobic nature of colloid surfaces acquired from stretching process, which in hydrophilic sand columns, dominated particle-surface charge interactions. Simulations using colloids with randomly distributed charge patches qualitatively produced the observed insensitivity in retention respecting aspect ratio under low charge coverage (<30%). Hence, particle shape influences were strongly coupled with colloid surface properties and flow hydrodynamics.

Hydrogels in the clinic.

Injectable hydrogels are one of the most widely investigated and versatile technologies for drug delivery and tissue engineering applications. Hydrogels' versatility arises from their tunable structure, which has been enabled by considerable advances in fields such as materials engineering, polymer science, and chemistry. Advances in these fields continue to lead to invention of new polymers, new approaches to crosslink polymers, new strategies to fabricate hydrogels, and new applications arising from hydrogels for improving healthcare. Various hydrogel technologies have received regulatory approval for healthcare applications ranging from cancer treatment to aesthetic corrections to spinal fusion. Beyond these applications, hydrogels are being studied in clinical settings for tissue regeneration, incontinence, and other applications. Here, we analyze the current clinical landscape of injectable hydrogel technologies, including hydrogels that have been clinically approved or are currently being investigated in clinical settings. We summarize our analysis to highlight key clinical areas that hydrogels have found sustained success in and further discuss challenges that may limit their future clinical translation.

Surface Modifications for Improved Delivery and Function of Therapeutic Bacteria.

Live therapeutic bacteria (LTBs) hold promise to treat microbiome-related diseases. However, few approaches to improve the colonization of LTBs in the gastrointestinal tract exist, despite colonization being a prerequisite for efficacy of many LTBs. Here, a modular platform to rapidly modify the surface of LTBs to enable receptor-specific interactions with target surfaces is reported. Inspired by bacterial adhesins that facilitate colonization, synthetic adhesins (SAs) are developed for LTBs in the form of antibodies conjugated to their surface. The SA platform is nontoxic, does not alter LTB growth kinetics, and can be used with any antibody or bacterial strain combination. By improving adhesion, SA-modified bacteria demonstrate enhanced in vitro pathogen exclusion from cell monolayers. In vivo kinetics of SA-modified LTBs is tracked in the feces and intestines of treated mice, demonstrating that SA-modified bacteria alter short-term intestinal transit and improve LTB colonization and pharmacokinetics. This platform enables rapid formation of an intestinal niche, leading to an increased maximum concentration and a 20% improvement in total LTB exposure. This work is the first application of traditional pharmacokinetic analysis to design and evaluate LTB drug delivery systems and provides a platform toward controlling adhesion, colonization, and efficacy of LTBs.